蔬菜土传病原菌三重PCR检测体系的建立与应用

doi:10.3864/j.issn.0578-1752.2019.12.005

中国农业科学 ›› 2019, Vol. 52 ›› Issue (12): 2069-2078.doi: 10.3864/j.issn.0578-1752.2019.12.005

蔬菜土传病原菌三重PCR检测体系的建立与应用

刘芮池^1,²,程有普²,柴阿丽¹(),石延霞¹,谢学文¹,帕提古丽³,李宝聚¹()

¹ 中国农业科学院蔬菜花卉研究所,北京 100081
² 天津农学院园艺园林学院,天津300384
³ 新疆农业科学院园艺作物研究所,乌鲁木齐 830091

收稿日期:2019-02-20 接受日期:2019-03-18 出版日期:2019-06-16 发布日期:2019-06-22
通讯作者: 柴阿丽,李宝聚
作者简介:刘芮池,E-mail： LIURUICHI0@163.com。
基金资助:
国家重点研发计划(2017YFD0200603);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS);农业部园艺作物生物学与种质创制重点实验室项目;自治区区域协同创新专项(2018E02027)

Establishment and Application of a Triplex PCR Detection System for Vegetable Soil-Borne Pathogens

LIU RuiChi^1,²,CHENG YouPu²,CHAI ALi¹(),SHI YanXia¹,XIE XueWen¹, PATIGULI³,LI BaoJu¹()

¹ Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081
² College of Horticultural and Landscape Architecture, Tianjin Agricultural University, Tianjin 300384
³ Institute of Horticulture, Xinjiang Academy of Agricultural Sciences, Urumqi 830091

Received:2019-02-20 Accepted:2019-03-18 Online:2019-06-16 Published:2019-06-22
Contact: ALi CHAI,BaoJu LI

摘要/Abstract

摘要：

【目的】针对生产中危害严重的3种蔬菜土传病原菌瓜果腐霉（Pythium aphanidermatum）、尖镰孢（Fusarium oxysporum）、大丽轮枝菌（Verticillium dahliae）,建立三重PCR（triplex PCR）检测体系,为蔬菜土传真菌病害的早期诊断和鉴定提供技术与方法。【方法】筛选3种病原菌特异性引物组合,通过设定不同的PCR退火温度、引物浓度、循环次数以及延伸时间,探索影响三重PCR扩增的因素,建立蔬菜土传病原菌三重PCR检测体系,并对体系灵敏度进行检测。为了验证建立体系的稳定性,选择2×Taq Master PCR Mix（北京博迈德生物技术有限公司）和TaKaRa Taq酶（大连宝生物工程有限公司）2个不同公司的试剂,分别利用C1000 Touch ^TM型（赛默飞世尔科技有限公司）与Aeris ^TM型（新加坡艺思高科技有限公司）热循环仪进行三重PCR反应,比较检测结果的可重复性。对田间采集的35份病害样本和149份土壤样本,分别进行三重PCR和病原菌分离检测,以确定所建立得三重PCR检测体系的适用性。【结果】三重PCR反应体系中引物对AsAPH2B/AsPyF、FOF1/FOR1、VActF/VActR可分别扩增出瓜果腐霉、尖镰孢、大丽轮枝菌长度为163、328和530 bp的特异性目的片段。反应体系（25 μL）：0.12 μmol·L ^-1 AsAPH2B/AsPyF,0.16 μmol·L ^-1 FOF1/FOR1,0.24 μmol·L ^-1 VActF/VActR,2×Taq Master PCR Mix 12.5 μL,退火温度为60.8℃,35个循环。该体系对病原菌纯培养物的检测灵敏度为10 ^-1ng·μL ^-1,对土壤中大丽轮枝菌、尖镰孢和瓜果腐霉的检测灵敏度分别为10 ⁵、10 ⁶个孢子/g土壤和10 ^-2mg菌丝/g土壤。分别采用2×Taq Master PCR Mix和TaKaRa Taq酶,利用C1000 Touch ^TM型和Aeris ^TM型热循环仪进行三重PCR,扩增结果一致,说明三重PCR体系检测稳定性好。对田间采集的病害和土壤样本进行检测,25份病害样本和71份土壤样本中检测出带菌,三重PCR检测与病原菌分离培养结果一致。【结论】本研究建立的瓜果腐霉、尖镰孢、大丽轮枝菌三重PCR检测体系具有灵敏度高、稳定性和重复性好的特点,能够快速、准确地检测田间病株及其根围土壤中的瓜果腐霉、尖镰孢、大丽轮枝菌,为蔬菜土传病害的早期预防和流行监测提供了有效的技术手段。

关键词: 蔬菜, 三重PCR, 土传病害, 瓜果腐霉, 尖镰孢, 大丽轮枝菌

Abstract:

【Objective】The objective of this study is to establish a triplex PCR system for the detection of Pythium aphanidermatum, Fusarium oxysporum and Verticillium dahliae, and to provide technology and method for early diagnosis and identification of soil-borne diseases of vegetables.【Method】Three sets of specific primers were selected to analyze the influencing factors of triplex PCR and the primer concentration, annealing temperature, amplification cycles and extension time of PCR reaction were optimized. The best triplex PCR detection system for vegetable soil-borne pathogenic fungi was established and the sensitivity of the system was detected. In order to test the stability of the system, 2×Taq Master PCR Mix (Beijing Biomed Co. Ltd) and TaKaRa Taq enzyme (TaKaRa Biotechnology Dalian Co. Ltd) were used by C1000 Touch ^TM (Thermo Fisher Scientific Co. Ltd) and Aeris ^TM type (Esco Micro Pte Ltd) thermal cycle meter for amplification. The triplex PCR and pathogen isolation and detection of 35 disease samples and 149 soil samples collected in the field were carried out to determine the applicability of the triplex PCR detection system.【Result】In the triplex PCR detection system, the specific target fragments of P. aphanidermatum, F. oxysporum and V. dahliae with the length of 163, 328 and 530 bp could be amplified by AsAPH2B/AsPyF, FOF1/FOR1, VActF/VActR, respectively. The 25 μL reaction system contained 0.12 μmol·L ^-1 AsAPH2B/AsPyF, 0.16 μmol·L ^-1 FOF1/FOR1, 0.24 μmol·L ^-1 VActF/VActR, 2×Taq Master PCR Mix 12.5 μL. The annealing temperature was 60.8℃ and the number of cycles was 35. The detection limitation was 10 ^-1ng·μL ^-1for pure culture of pathogens. For artificially infected substrate, the detection limitations were 10 ⁵, 10 ⁶ spores/g and 10 ^-2 mg hyphae/g for V. dahliae, F. oxysporum and P. aphanidermatum, respectively. The results were consistent of 2×Taq Master PCR Mix and TaKaRa Taq enzyme, which were amplified by C1000 Touch ^TM and Aeris ^TM type thermal cycle meter. It meant that the triplex PCR system was stable. The infected tissues and soil samples collected in the field were detected, and 25 tissues and 71 soil samples were detected for carrying pathogen, which were consistent with the results of isolation and culture.【Conclusion】The triplex PCR detection system established in this study has the characteristics of high sensitivity, stability and reproducibility, and can be used for rapid and accurate detection of P. aphanidermatum, F. oxysporum and V. dahliae in the infected plant and the surrounding soil. It provides an effective technical means for early prevention and epidemic monitoring of vegetable soil-borne diseases.

Key words: vegetable, triplex PCR, soil-borne disease, Pythium aphanidermatum, Fusarium oxysporum, Verticillium dahliae

刘芮池,程有普,柴阿丽,石延霞,谢学文,帕提古丽,李宝聚. 蔬菜土传病原菌三重PCR检测体系的建立与应用[J]. 中国农业科学, 2019, 52(12): 2069-2078.

LIU RuiChi,CHENG YouPu,CHAI ALi,SHI YanXia,XIE XueWen, PATIGULI,LI BaoJu. Establishment and Application of a Triplex PCR Detection System for Vegetable Soil-Borne Pathogens[J]. Scientia Agricultura Sinica, 2019, 52(12): 2069-2078.

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不同地区病害样本三重PCR检测"

采样时间 Sampling time	采样地点 Sampling site	寄主 Host	疑似病害 Suspected disease	采样部位 Sampling position	三重PCR Triple PCR	分离培养 Isolated culture	样本数量 Number of samples
2017-12-09	浙江苍南 Cangnan, Zhejiang	茄子Eggplant	黄萎病Verticillium wilt	茎基Stem	+	V. dahliae	4
				土壤Soil	+	V. dahliae	4
2018-01-16	山东寿光 Shouguang, Shandong	黄瓜Cucumber	枯萎病Fusarium wilt	茎基Stem	+	F. oxysporum	1
				土壤Soil	+	F. oxysporum	1
2018-03-15	浙江苍南 Cangnan, Zhejiang	黄瓜Cucumber	枯萎病Fusarium wilt	茎基Stem	+	F. oxysporum	1
				土壤Soil	+	F. oxysporum	1
2018-03-20	山东寿光 Shouguang, Shandong	黄瓜Cucumber	根腐病Root rot	茎基Stem	+	P. aphanidermatum	2
				土壤Soil	+	P. aphanidermatum	2
2018	浙江苍南 Cangnan, Zhejiang	黄瓜Cucumber	枯萎病Fusarium wilt	茎基Stem	+	F. oxysporum	2
				土壤Soil	+	F. oxysporum	2
2018-10-21	山东寿光 Shouguang, Shandong	黄瓜Cucumber	根腐病Root rot	茎基Stem	+	P. aphanidermatum	1
				土壤Soil	+	P. aphanidermatum	1
2018-10-21	山东寿光 Shouguang, Shandong	黄瓜Cucumber	根腐病Root rot	茎基Stem	-	—	1
				土壤Soil	-	—	1
2018-03-15	浙江苍南 Cangnan, Zhejiang	黄瓜Cucumber	枯萎病Fusarium wilt	茎基Stem	-	—	1
				土壤Soil	-	—	1
2018-03-20	山东寿光 Shouguang, Shandong	黄瓜Cucumber	根腐病Root rot	茎基Stem	-	—	1
				土壤Soil	-	—	1
2018-08-31	新疆喀什 Kashi, Xinjiang	甜瓜Muskmelon	枯萎病Fusarium wilt	茎基Stem		F. oxysporum	11
				茎基Stem	-	—	4
2019-01-02	辽宁绥中 Suizhong, Liaoning	黄瓜Cucumber	枯萎病Fusarium wilt	茎基Stem	+	F. oxysporum	3
				茎基Stem	-	—	3
2017-2018	浙江苍南 Cangnan, Zhejiang	—	—	土壤Soil	-	—	48
				土壤Soil	+	F. oxysporum	12
				土壤Soil	+	V. dahliae	12
2018	山东寿光 Shouguang, Shandong	—	—	土壤Soil	-	—	27
				土壤Soil	+	V. dahliae	3
				土壤Soil	+	F. oxysporum	6
				土壤Soil	+	P. aphanidermatum	12
2018-08-24	宁夏平罗 Pingluo, Ningxia	—	—	土壤Soil	+	P. aphanidermatum	9
2018-08-24	浙江永康 Yongkang, Zhejiang	—	—	土壤Soil	+	P. aphanidermatum	6

表4

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病原菌 Pathogen	寄主 Host	菌株编号 Strain number	引物 Primer
病原菌 Pathogen	寄主 Host	菌株编号 Strain number	AsAPH2B/AsPyF	FOF1/FOR1	VActF/VActR
瓜果腐霉P. aphanidermatum	番茄Tomato	FQ15092209	+	-	-
尖镰孢F. oxysporum	黄瓜Cucumber	HG12090201	-	+	-
大丽轮枝菌V. dahliae	茄子Eggplant	QZ12061103	-	-	+
茄镰孢F. solani	黄瓜Cucumber	HG14060601	-	-	-
茄镰孢F. solani	芹菜Celery	QC15061008	-	-	-
核盘菌Sclerotinia sclerotiorum	青花菜Broccoli	QHC18032301	-	-	-
核盘菌Sclerotinia sclerotiorum	茄子Eggplant	QZ11102201	-	-	-
辣椒疫霉Phytophthora capsici	辣椒Pepper	LJ12010805	-	-	-
辣椒疫霉Phytophthora capsici	辣椒Pepper	LJ18081001	-	-	-
灰葡萄孢Botrytis cinerea	番茄Tomato	FQ11051301	-	-	-
灰葡萄孢Botrytis cinerea	黄瓜Cucumber	HG11021509	-	-	-
立枯丝核菌Rhizoctonia solani	白菜Chinese cabbage	BC16052001	-	-	-
立枯丝核菌Rhizoctonia solani	生菜Lettuce	SC16052001	-	-	-
茄科劳尔氏菌Ralstonia solanacearum	番茄Tomato	FQ12080802	-	-	-
茄科劳尔氏菌Ralstonia solanacearum	番茄Tomato	FQ16060706	-	-	-
胡萝卜果胶杆菌Pectobacterterium carotovorum	黄瓜Cucumber	HG1501503604	-	-	-
胡萝卜果胶杆菌Pectobacterterium carotovorum	白菜Chinese cabbage	BC18082306	-	-	-
密执安棒形杆菌密执安亚种 Clavibacter michiganensis subsp. michiganensis	番茄Tomato	FQ18082408	-	-	-
	番茄Tomato	FQ18082410	-	-	-

病原菌Pathogen	序列Sequence (5′-3′)	产物大小Product length (bp)	参考文献Reference
瓜果腐霉 P. aphanidermatum	AsAPH2B: GCGCGTTGTTCACAATAAATTGC	163	[19]
瓜果腐霉 P. aphanidermatum	AsPyF: CTGTTCTTTCCTTGAGGTG	163	[19]
尖镰孢F. oxysporum	FOF1: ACATACCACTTGTTGCCTCG	328	[20]
尖镰孢F. oxysporum	FOR1: CGCCAATCAATTTGAGGAACG	328	[20]
大丽轮枝菌V. dahliae	VActF: TAATTCACAATGGAGGGTAGG	530	[21]
大丽轮枝菌V. dahliae	VActR: GTAAGGATACCACGCTTGG	530	[21]

病原菌Pathogen	GenBank登录号 GenBank accession number	同源性Homology rate (Blastn) (%)
瓜果腐霉P. aphanidermatum	KU211462.1	99
尖镰孢F. oxysporum	KU528856.1	99
大丽轮枝菌V. dahliae	KU057923.1	99

蔬菜土传病原菌三重PCR检测体系的建立与应用

Establishment and Application of a Triplex PCR Detection System for Vegetable Soil-Borne Pathogens

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